1. Field of the Invention
This invention relates to a rapid immunoassay kit and method for semi-quantitatively detecting antibodies in human saliva to antigens of disease-related microorganisms, e.g., antibodies to Mycobacterium tuberculosis. This invention also encompasses an alternative embodiment that permits quantitative, though less rapid, detection of antibodies in saliva by adapting the methodology of the semi-quantitative immunoassay to an enzyme linked immunosorbant assay (ELISA). Within this invention, this alternative embodiment is referred to as the quantitative immunoassay, or similar, to distinguish it from the rapid, semi-quantitative immunoassay.
2. Description of the Prior Art
Though not substantially related to the invention described herein, there have been several efforts of peripheral interest. Ebersole has described a SEROLOGICAL METHOD FOR THE IDENTIFICATION OF MICROORGANISMS in U.S. Pat. No. 4,458,014 for the identification of diseases of the mouth. Chen et al. have described in U.S. Pat. No. 4,866,167 a DETECTION OF HUMAN ORAL CELLS BY NUCLEIC ACID HYBRIDIZATION to detect oral bacterial species. The methods of both Ebersole and Chen et al. are technically complex, time consuming, not rapid and are not based on detecting antibodies in saliva to antigens of disease-related microorganisms.
Olson et al. have described an IMMUNOLOGICAL COLOR CHANGE TEST INVOLVING TWO DIFFERENTLY COLORED REAGENT SPOTS in U.S. Pat. No. 4,639,419. Their patent describes a substantially different methodology than that described herein. This test is an agglutination reaction directed toward identifying antigenic material wherein a colored substrate and colored reagent combine, in positive reactions, to give the appearance of a third color.
Higerd and Goust have described an IMMUNOSUPPRESSIVE EXTRACELLULAR PRODUCT FROM ORAL BACTERIA in U.S. Pat. No. 4,268,434. Their patent relates to a method of producing an extracellular immunosuppressive bacterial material from various bacteria to suppress the natural immunity in patients where this outcome is desired, e.g., organ transplant patients. This procedure has substantially different objectives and methodology than the invention described herein.
Antibodies are naturally produced biomolecules which react specifically with usually foreign biomolecules called antigens. Disease-related microbial infections, e.g., Mycobacterium tuberculosis which causes tuberculosis, are usually characterized by the production of antibodies to the specific antigens of disease-related microorganisms. Antibodies are also produced with other diseases and afflictions, e.g., autoimmune diseases where there is an often destructive antibody response to the hostxe2x80x94not necessarily related to a microbial antigen. In the case of autoimmune diseases, the host usually supplies the antigens of disease-related microorganisms. Within this invention, the term xe2x80x9cdisease-related antigensxe2x80x9d includes microbial antigens and other substances capable of possessing antigenic properties and which are associated with specific diseases, conditions and disorders, including infectious diseases and autoimmune diseases. Antibodies are expressed in saliva; their detection in saliva is fundamental and unique to this invention.
This invention, as an example, can determine individuals actively or previously infected with Mycobacterium tuberculosis and thus aid in the diagnosis of tuberculosis. Mycobacterium tuberculosis causes tuberculosis (1-4) and this is widely acknowledged throughout the medical community. There are several screening tests for tuberculosis. The Mantoux test uses tuberculin purified protein derivative (PPD) which is injected intracutaneously (e.g., Tubersol(copyright), Connaught Laboratories Limited, Willowdale, Ontario, Canada) (1). A delayed hypersensitivity reaction develops in individuals having previous infection with Mycobacterium tuberculosis. The injection site is normally read within 48 to 72 hours after intracutaneous injection of the antigen; a palpable induration measuring 10 mm in diameter or more is considered a positive reaction. This procedure is accepted as an aid in the diagnosis of tuberculosis infection.
The Heaf test uses a multiple puncture disk which introduces needles through concentrated Old Tuberculin applied to the skin (1). The tine test uses tuberculin adhering to metal tines; inoculation is accomplished by simple pressure into the skin (1). The Heaf and tine tests are acceptable for screening but should be confirmed by the Mantoux test (1). Antigenic material can also be applied by scratch, i.e., Pirquet""s test (2). Similar to the Mantoux test, these tests generally require 48 to 72 hours after inoculation before results can be determined. The Bacillus of Caimette and Guerin (BCG) is a live, attenuated strain of Mycobacterium bovis which has been used with varying success as a vaccine against tuberculosis in countries where the prevalence of tuberculosis is high (5). Mycobacterium bovis is not normally found in humans, but since it shares antigens present in Mycobacterium tuberculosis, it can serve as an antigen source to detect host antibodies to Mycobacterium tuberculosis. BCG causes tuberculin conversion to positive; it has also been used to stimulate the immune system against a variety of medical conditions. In this invention, both PPD and BCG can serve as suitable antigens to detect host antibodies to desired mycobacteria.
Other antigens have been described. Maes has described A60-ANTIGEN FROM MYCOBACTERIA AND USE THEREOF AS TUBERCULIN VACCINE in U.S. Pat. No. 4,965,192. This patent describes the A60-antigen as effective for detecting prior exposure of an individual to mycobacterial infections through the use of a cutaneous test. This patent is similar to other inoculation tests mentioned earlier except that a new antigen is used and 24 to 48 hours are required to observe the responses at the test site.
Mycobacterium tuberculosis whole cells (inactivated), lipoarabinomannan of Mycobacterium tuberculosis (6-8) and other mycobacterial derivatives can serve as antigen sources to detect host antibodies to mycobacteria in this invention.
To continue with the Mycobacterium tuberculosis example of this invention, a major advantage is that tuberculosis screening can be done rapidlyxe2x80x94in approximately 5 minutesxe2x80x94in one visit and in a non-invasive manner. The advantages of this invention are significant when compared with earlier tests that are invasive, take 48 to 72 hours to obtain results and require two visits of the subject, e.g., the Mantoux and other tuberculosis screening tests. These earlier tests are considered too slow and are invasive. Similar limitations apply to other medical screening and diagnostic tests that are not rapid, invasive (e.g., require blood or serum samples) or involve culturing or other complicated and expensive laboratory procedures. The premise of this use of the assay is that individuals infected with Mycobacterium tuberculosis develop antibodies to this bacterial species which are present in their saliva and which react with mycobacterial antigens. The antibodies are then labeled and color development detected and read visually after addition of an appropriate enzymatic substrate, if required. Color development signifies positive individuals and permits semi-quantitative assessment of antibody levels. Active or previous infection with Mycobacterium tuberculosis is, therefore, determined. This assay aids in the diagnosis of tuberculosis and is rapid, non-invasive, uncomplicated and inexpensive. Less rapid but quantitative simultaneous assessment of multiple (or single) saliva samples is accomplished by adapting the semi-quantitative assay to an ELISA.
What is needed is a rapid, simple, non-invasive assay to semi-quantitatively detect antibodies in saliva to antigens of disease-related microorganisms, e.g., antibodies to Mycobacterium tuberculosis that react with mycobacterial antigens. This assay uses human saliva, is non-invasive, can be developed and read in less than an hour, preferably in about 5 minutes, and is technically simple to operate. A rapid immunoassay to semi-quantitatively detect antibodies in saliva to antigens of disease-related microorganisms, is unique and has never been reported.
What is also needed is an immunoassay capable of quantitatively assessing multiple saliva samples simultaneously. A quantitative immunoassay is needed in instances where specific quantitative measurements or the ability to assess multiple samples simultaneously are desired over the need for a more rapid, semi-quantitative assessment. The adaptation of the rapid, semi-quantitative assay to an ELISA to quantitatively assess a single saliva sample or multiple saliva samples simultaneously is unique and has never been reported.
Accordingly, an object of this invention is an immunodiagnostic assay kit and method to rapidly and semi-quantitatively detect antibodies in saliva produced as part of an immunological response to specific, antigens of disease-related microorganisms, i.e., a host antibody response.
An additional object of this invention is a device for conducting the rapid and semi-quantitative inmmunoassay.
A further object of this invention is the adaptation of the rapid immunoassay to an ELISA permitting simultaneous, quantitative detection of antibodies in multiple (or single) saliva samples produced as part of an immunological response to specific disease-related antigens, i.e., a host antibody response.
These and additional objects of the invention are accomplished by an immunoassay kit and method for rapidly and semi-quantitatively detecting antibodies in saliva to antigens of disease-related microorganisms, and by adaptation of the rapid, semi-quantitative immunoassay to an ELISA thereby permitting quantitative assessment of a single saliva sample or simultaneous quantitative assessment of multiple saliva samples. With this invention, the semi-quantitative assay can be performed on an aliquot of a saliva sample. The semi-quantitative assessment can then be extended by using the quantitative assay to assess a different aliquot of the same saliva sample.
Antigens of disease-related microorganisms are immobilized on a solid substrate and contacted with a saliva sample from the human subject being tested. The saliva samples are filtered with a sample filter or treated with some other separating device such as a centrifuge prior to their contact with the immobilized antigens. Antibodies to the antigens may be present in the saliva sample. These primary antibodies, if present, bind to the immobilized antigens. After blocking, the primary antibodies are then contacted typically with secondary antibodies specific for the primary antibodies having a label or indicator capable of being detected, e.g., alkaline phosphatase. Secondary antibodies can be anti-human IgG, IgA, IgM, alone or in combination. After the addition of an appropriate enzymatic substrate, if required, the label develops identifying the presence of the antibodies whereby active or previous infection with the antigen, e.g., Mycobacterium tuberculosis, is determined. The device for conducting the semi-quantitative assay is a frame or support which holds a solid substrate capable of immobilizing the antigens of interest while permitting drainage of other materials or fluids away from the bound antigens. The device for the quantitative assay includes an ELISA plate reader, 96-well plates, a plate washer, a multidrop dispenser, and related ELISA equipment. The 96-well plates, or similar, are capable of immobilizing the desired antigens of disease-related microorganisms thereby allowing the fundamental immunologic reaction of the semi-quantitative assay to take place in the wells of the plates and measured quantitatively with the ELISA plate reader. For the quantitative assay, each well of the 96-well plates serves as a solid substrate capable of immobilizing the desired antigens of disease-related microorganisms.